Junctional Adhesion Molecule a (JAM-A) Associates with CD36 upon Platelet Activation and Cytoskeleton in a Phosphorylation-Dependent Manner to Promote Platelet-Platelet Contacts

Background: Platelets play a fundamental role inmaintaining hemostasis. Stability of hemostatic plug is crucial for arresting blood loss during vascular breach. JAM-A is a member of the immunoglobulin superfamily that restricts α _IIbβ ₃ to its low affinity state by recruiting CSK to the α _IIbβ ₃-c-Src complex in resting platelets. Agonist-induced inside-out signaling causes JAM-A to dissociate from the α _IIbβ ₃-Src complex, thus facilitating activation of α _IIbβ ₃ and outside-in signaling. However, the function of JAM-A in the process of hemostasis is poorly understood.

Aim: To evaluate the role of JAM-A in activated platelets and on hemostasis.

Methods: Washed human platelets were used in these studies. Full-length cytoplasmic domain of JAM-A (cyto JAM-A) and JAM-A cytoplasmic domain lacking C-terminal PDZ-domain-binding motif (ΔPDZ cyto JAM-A) were expressed as GST-fusion proteins in bacteria using pGEX4T-1 vector. GST-fusion proteins were purified using glutathione beads. Pull-down assays were performed by incubating glutathione GST-fusion proteins beads with lysates of washed human platelets (4×10 ⁸/mL). GST beads alone without the fusion proteins were used as a control. Immunoprecipitation studies were performed using washed human platelets (4x10 ⁸/mL) lysed with CHAPS lysis buffer and precleared by using Protein G Sepharose beads. Specific primary antibodies and isotype specific IgGs (control) were used for immunoprecipitation followed by western blotting. To identify protein complexes two-dimensional blue-native polyacrylamide gel electrophoresis was performed in which protein complexes were first separated based on their size (1 ^st dimension) and individual proteins in each complex were separated using SDS PAGE (2 ^nd dimension). JAM-A S ²⁸⁴ phosphorylation was detected using anti-Phospho-S ²⁸⁴. Rap1 activation assay was performed by pull-down GTP-bound Rap1 following the manufacturer's instructions.

Results: CD9 and α _IIbβ ₃ co-immunoprecipitated with JAM-A from resting platelet lysates suggesting that CD9 is a part of the JAM-A/α _IIbβ ₃ complex. In a pull-down assay recombinant full-length JAM-A cytoplasmic tail fused with GST, but not JAM-A tail lacking the PDZ domain motif fused with GST pull-down CD9 and α _IIbβ ₃ from resting platelet lysates suggesting that PDZ-domain binding motif of JAM-A is responsible for its interaction with CD9/α _IIbβ ₃ complex. Interestingly, platelet activation resulted in the dissociation of JAM-A from the CD9/α _IIbβ ₃ complex as seen by loss of interaction between JAM-A and CD9/α _IIbβ ₃ in co-immunoprecipitation and blue-native PAGE assays. It is reported that platelet activation results in rapid phosphorylation of JAM-A on S ²⁸⁴ in a PKC-dependent manner. We found that S ²⁸⁴ phosphorylated JAM-A associates with CD36 and this complex moves to the Triton X-100 insoluble cytoskeletal fraction upon platelet activation suggesting that phosphorylation of JAM-A may assist its localization to cytoskeleton. Interestingly, we found that induction of conformational change in α _IIbβ ₃ using Mn ²⁺ (10μM) activates JAM-A S ²⁸⁴ phosphorylation. Furthermore, Mn ²⁺ also induced a robust activation of Rap1, a key signaling molecule involved in junctional assembly. Both JAM-A phosphorylation and Rap1 activation were significantly ( P<0.05) inhibited by a pan PKC inhibitor, bisindolylmaleimide (10μM) suggesting that S ²⁸⁴ phosphorylated JAM-A/CD36 complex may support junctional assembly within the platelet plug.

Conclusion: JAM-A, through its cytoplasmic domain, associates with α _IIbβ ₃ and CD9 in resting platelets. Upon platelet activation JAM-A rapidly dissociates from this complex and is phosphorylated on S ²⁸⁴. Phosphorylated JAM-A then associates with CD36 and cytoskeleton leading to activation of Rap1, a protein important in junctional assembly between platelets within a hemostatic plug.